ORIGINAL ARTICLE

Genetic analysis of avian influenza A viruses isolatedfrom domestic waterfowl in live-bird markets of Hanoi, Vietnam,preceding fatal H5N1 human infections in 2004

Samadhan J. Jadhao Æ Doan C. Nguyen Æ Timothy M. Uyeki Æ Michael Shaw Æ Taronna Maines ÆThomas Rowe Æ Catherine Smith Æ Lien P. T. Huynh Æ Ha K. Nghiem Æ Diep H. T. Nguyen Æ Hang K. L. Nguyen ÆHanh H. T. Nguyen Æ Long T. Hoang Æ Tung Nguyen Æ Lien S. Phuong Æ Alexander Klimov Æ Terrence M. Tumpey ÆNancy J. Cox Æ Ruben O. Donis Æ Yumiko Matsuoka Æ Jacqueline M. Katz

Received: 19 January 2009 / Accepted: 9 June 2009 / Published online: 4 July 2009

� US Government 2009

Abstract The first known cases of human infection with

highly pathogenic avian influenza (HPAI) H5N1 viruses in

Vietnam occurred in late 2003. However, HPAI H5N1 and

low-pathogenic avian influenza (LPAI) H5N2 and H9N3

viruses were isolated from domestic waterfowl during live-

bird market (LBM) surveillance in Vietnam in 2001 and

2003. To understand the possible role of these early viruses

in the genesis of H5N1 strains infecting people, we per-

formed sequencing and molecular characterization. Phylo-

genetic analysis revealed that the hemagglutinin (HA)

genes of two geese HPAI H5N1 strains belonged to clade 3,

and their surface glycoprotein and replication complex

genes were most closely related (98.5–99.7% homologous)

to A/duck/Guangxi/22/01 (H5N1) virus, detected contem-

porarily in southern China, whilst the M and NS genes were

derived from an A/duck/Hong Kong/2986.1/00 (H5N1)-like

virus. The H5 HA gene of the duck HPAI H5N1 strain

belonged to clade 5 and acquired a gene constellation from

A/quail/Shantou/3846/02 (H5N1), A/teal/China/2978.1/02

(H5N1) and A/partridge/Shantou/2286/03 (H5N1)-like

viruses. The phylogenetic analysis further indicated that all

eight gene segments of goose and duck HPAI H5N1 and

LPAI H5N2 viruses were distinct from those of H5N1

clade-1 viruses known to have caused fatal human infec-

tions in Vietnam since late 2003. The duck H9N3 isolates

derived genes from aquatic-bird influenza viruses, and their

H9 HA belonged to the Korean lineage. The PB2 gene of A/

duck/Vietnam/340/01 (H9N3) virus had lysine at position

627. Based on the molecular characterization of specific

amino acid residues in the surface and relevant internal

S. J. Jadhao � D. C. Nguyen � T. M. Uyeki � M. Shaw �T. Maines � T. Rowe � C. Smith � A. Klimov �T. M. Tumpey � N. J. Cox � R. O. Donis (&) �Y. Matsuoka � J. M. Katz

Influenza Division, MS-G16, National Center for Immunization

and Respiratory Diseases, Centers for Disease Control and

Prevention, 1600 Clifton Road, NE, Atlanta, GA 30333, USA

e-mail: RDonis@cdc.gov

D. C. Nguyen � L. P. T. Huynh � H. K. Nghiem �H. K. L. Nguyen � H. H. T. Nguyen � L. T. Hoang

National Institute of Hygiene and Epidemiology, Hanoi,

Vietnam

D. H. T. Nguyen � T. Nguyen � L. S. Phuong

National Center for Veterinary Diagnosis, Hanoi, Vietnam

Present Address:S. J. Jadhao

Southeast Poultry Research Laboratory, USDA-ARS,

934 College Station Road, Athens, GA 30605, USA

e-mail: Samadhan.Jadhao@ars.usda.gov

Present Address:D. C. Nguyen

Department of Biology, Georgia State University,

Atlanta, GA 30302, USA

Present Address:T. Rowe

Department of Immunology, University of Toronto, Toronto,

ON, Canada

Present Address:Y. Matsuoka

Laboratory of Infectious Diseases, National Institute of Allergy

and Infectious Diseases, National Institute of Health, Bethesda,

MD 20892, USA

123

Arch Virol (2009) 154:1249–1261

DOI 10.1007/s00705-009-0429-2

protein-coding genes, the Vietnamese H5N1 and H9N3

virus isolates indicated specificity to avian cell surface

receptor and susceptibility for currently licensed anti-

influenza A virus chemotherapeutics. Our findings suggest

that the H5N1 and H5N2 viruses that circulated among

geese and ducks in LBMs in Hanoi, Vietnam, during 2001

and 2003 were not the immediate ancestors of the clade-1

viruses associated with fatal human infections in Vietnam.

The clade-1 HPAI H5N1 viruses were independently

introduced into Vietnam.

Introduction

Widespread outbreaks of highly pathogenic avian influenza

(HPAI) H5N1 viruses among poultry in Vietnam began in

late 2003 [1, 20]. HPAI H5N1 outbreaks were also reported

among domestic poultry and migratory birds in Asian,

African and some European countries during subsequent

years [1, 2, 9, 18–20, 26, 28, 36, 38]. From November 2003

through June 2008, HPAI H5N1 viruses caused fatal

human infections in 257 of 418 confirmed cases in 15

countries in Asia, Africa, the Middle East, and Europe [39].

The origins of the HA genes of all viruses isolated from

humans has been traced to domestic poultry in southern

China [1, 8, 12, 20, 32]. A limited number of cloacal and

tracheal swabs were collected from domestic waterfowl in

the course of epidemiological study of avian influenza A

viruses in live bird markets (LBMs) in Hanoi, Vietnam, in

2001 [24]. On the basis of this pilot surveillance in LBMs,

we reported isolation of HPAI H5N1, low-pathogenic

avian influenza (LPAI) H5N2, H4N6 and H9N3 viruses

from apparently healthy geese and ducks [24]. The H5N1

avian influenza viruses from geese and ducks possessed

polybasic amino acid sequence at the HA cleavage site.

One of the goose H5N1 isolates was tested and proven

highly pathogenic in a chicken intravenous pathotyping

experiment based on the criteria of Office International des

Epizooties, World Organization for Animal Health [24].

Concurrent circulation of several genotypes of HPAI H5N1

viruses was reported among domestic ducks between 1999

and 2002 in southern China [6]. Since late 2003, HPAI

H5N1 outbreaks among domestic poultry in Vietnam have

resulted in zoonotic transmission and fatal human infec-

tions in 56 of 111 cases to date [39]. A partial nucleotide-

sequence-based phylogeny of the HA and NA genes of the

goose H5N1, duck H5N2 and duck H9N3 avian influenza

viruses isolated in 2001 in the LBMs of Hanoi, Vietnam,

was reported previously [24]. However, information on the

origin of the internal genes of these avian influenza virus

isolates and complete HA and NA nucleotide sequence

data were lacking. Phylogenetic and molecular analysis of

these H5N1 and H9N3 avian influenza viruses isolated in

2001 in the LBMs of Hanoi, Vietnam, could enhance our

understanding about the origin of the H5N1 viruses from

late 2003 and subsequent outbreaks in poultry and fatal

H5N1 human infections in Vietnam. In this study, we

investigated whether the HPAI H5N1 viruses isolated in

2001 and 2003 from domestic waterfowl in LBMs of Ha-

noi, Vietnam, were the precursors of the HPAI H5N1

viruses that caused fatal human infections in 2003. Phylo-

genetic analysis of the LPAI H5N2 and H9N3 viruses

was performed to understand if these viruses contributed

genes to the HPAI H5N1 viruses that caused fatal human

infections after late 2003. We found that the HPAI H5N1

viruses isolated from domestic waterfowl in 2001 and 2003

were not the closest common ancestors of the clade-1 viruses

that caused human infections in Vietnam in late 2003–2004.

The clade-1 H5N1 viruses were independently introduced

into Vietnam.

Materials and methods

Avian influenza viruses

The avian influenza A virus strains isolated from geese and

ducks at LBMs in Hanoi, Northern Vietnam, or during

routine surveillance in domestic ducks are shown in

Table 1. Viruses were propagated in 10-day-old embryo-

nated chicken eggs. RNA was extracted from allantoic

fluids using a commercial kit per manufacturer instructions

(Qiagen Inc., Valencia, CA). All research with these

Vietnamese avian influenza viruses was conducted under

biosafety level 3 containment, including enhancements

required by the US Department of Agriculture and the

Select Agent Program (see interim guidance at http://www.

cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm).

Nucleotide sequencing and phylogenetic analysis

Influenza virus genes were amplified with segment-specific

oligonucleotides using the One-Step RT-PCR kit (Qiagen

Inc., Valencia, CA) [16]. The nucleotide sequences were

determined on an automated Applied Biosystems ABI 3100

sequencer using a BigDye terminator v3.1 cycle sequenc-

ing kit (Applied Biosystems Inc. Foster City, CA).

Sequence outputs were analyzed with the SeqMan module

of the Lasergene software (DNAStar Inc., Madison, WI).

Nucleotide sequences were aligned by the Clustal W

algorithm using the MegAlign 5.07 module in Lasergene

molecular biology software (DNAStar Inc., Madison, WI).

The influenza virus gene nucleotide sequences have been

submitted to the Global Initiative on Sharing Avian Influ-

enza Data (GISAID) database [3], and the sequence

accession numbers EPI168444 through EPI168481 can be

1250 S. J. Jadhao et al.

123

accessed at http://platform.gisaid.org. Phylogenetic analy-

sis was performed by the neighbor-joining method with the

maximum composite likelihood model and 10,000 boot-

strap replicate analyses using MEGA 4.0 software [34].

The sequence regions used for phylogenetic analysis of

various gene segments were based on the availability of the

reference virus sequences in GenBank: PB2 (1029-2135),

PB1 (92-1408), PA (52-2151), H5 HA (42-1640), NP (133-

1034), N1 NA (63-1073), M (32-782), NS (78-719), H9

HA (148-1551), N2 NA (89-1271), N3 NA (19-1449). The

phylogenetic trees for PB2, N1 NA, NS, H9 HA, N2 NA

and N3 NA genes are not included in this report but are

available upon request. The analysis and assignment of

genotypes to the influenza virus isolates was performed

using a web tool as described previously [21]. Viruses

representing relevant HA clades were included as a point of

reference in the phylogenetic tree in Fig. 1 [11].

Molecular analysis

H5 HA amino acid sequences comparison of all H5N1

viruses known to have infected people between the years

1997 and 2005 were aligned using the Clustal W algorithm

(DNAStar Inc., Madison, WI). The highest nucleotide/

amino acid identities to the published influenza A virus

sequences were determined using FluGenome BLAST

search [21].

Results and discussion

Phylogenetic and genotype analyses

The HA gene of Vietnamese H5N1 viruses of goose origin

from 2001 (isolates Gs113 and Gs324, Table 1) were

closely related to each other (Fig. 1a; Table 2) [24]. Six of

their genes, including ones coding for basic polymerase 2

(PB2), basic polymerase 1 (PB1), acidic polymerase (PA),

HA, nucleoprotein (NP), and neuraminidase (NA), were

derived from an A/duck/Guangxi/22/01-like H5N1 virus,

as indicated by the high nucleotide sequence homology

values, ranging from 98.5 to 99.7% (Table 2). The matrix

(M) genes of the goose H5N1 viruses were derived from an

A/duck/Hong Kong/2986.1/00-like H5N1 virus (99.4%

homology) (Fig. 1e; Table 2). The progenitor of the non-

structural (NS) genes of the goose viruses was the A/duck/

Hong Kong/380.5/01-like (H5N1) virus, which showed

98.1% homology (Table 2). A/duck/Guangxi/22/01-like

H5N1 viruses were reported to circulate among domestic

ducks in southern China during 1999–2002 [6]. These

phylogeny findings indicate that the Vietnamese goose-

origin H5N1 isolates Gs113 and Gs324 have undergone

reassortment with A/duck/Guangxi/22/01-like (H5N1)Ta

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H5N1 avian influenza viruses from Vietnam 1251

123

A/duck/Hong Kong/205/77A/duck/Hong Kong/698/79

A/duck/Potsdam/22164/84A/duck/Mongolia/54/01

A/duck/Mongolia/500/0186

84 Early aquatic bird isolates

A/duck/Vietnam/342/01 (H5N2)A/duck/Primoriae/2633/01

A/duck/Hokkaidio/101/04A/mallard/Sweden/58/02

A/goose/Guangdong/1/96A/Hong Kong/156/97

A/duck/Anyang/AVL-1/01A/silky chicken/Hong Kong/YU100/02

A/ hi k /H K /FY150/01

8299

100

100100

81

Recent aquatic bird isolates

Clade 0

A/chicken/Hong Kong/FY150/01A/duck/Hong Kong/2986.1/00A/duck/Fujian/17/01A/goose/Hong Kong/3014.5/00

A/duck/Guangxi/22/01A/goose/Vietnam/113/01 (H5N1)A/goose/Vietnam/324/01 (H5N1)

A/chicken/HongKong/YU822.2/01A/ hi k /H K /8791/01

100

99

9993

Clade 3

A/chicken/Hong Kong/8791/01A/goose/Guiyang/337/06A/chicken/Guiyang/846/06

A/chicken/Shanxi/2/06A/teal/China/2978.1/02A/quail/Shantou/3846/02

A/duck/Vietnam/NCVD1/03 (H5N1)A/chicken/Vietnam/Ncvd8/03

A/goose/Guangxi/914/04

100

100

98

98

9486

0.01

Clade 4

Clade 7

Clade 5

A/goose/Guangxi/914/04A/chicken/Guangxi/2439/04

A/duck/Zhejiang/52/00A/swine/Fujian/F1/01A/swine/Fujian/1/03A/duck/Hubeiwg/02/6

A/chicken/Jilin/9/04A/duck/Guangxi/2775/05

A/chicken/Hong Kong/YU22/02

100

98

94

84

86

Cl d 8

Clade 6

Clade 9A/chicken/Hong Kong/YU22/02

A/chicken/Henan12/04A/mallard/Guangxi/wt/04

A/duck/China/E319-2/03A/goose/Guangxi/3316/05A/duck/Hunan/139/05

A/duck/Guiyang/3009/05A/Anuhai/1/05A/ bh goose/Qinghai/12/05

86

91

9760

82

Clade 2

Clade 8

A/ bh goose/Qinghai/12/05A/chicken/Indonesia/7/03

A/duck/Indonesia/MS/04A/Indonesia/283H/06

A/goose/Hong Kong/739.2.02A/Hong Kong/213/03

A/duck/Yunnan/215/03A/chicken/Vietnam/27/03A/chicken/Vietnam/C58/04

9899

68

98

85

A/quail/Vietnam/036/04A/Vietnam/3062/04 (H5N1)

A/Vietnam/1194/04 (H5N1)A/Vietnam/1203/04 (H5N1)

A/duck/Vietnam/NCVD07/0590

90 Clade 1

A: H5 HA

1252 S. J. Jadhao et al.

123

virus to acquire surface glycoprotein genes and four repli-

cation complex genes. The matrix and NS segments were

acquired from an A/duck/Hong Kong/380.5/01-like (H5N1)

virus. The reassortment events were likely occurred in

southern China before introduction of these viruses in geese

and their detection in the LBMs of Hanoi, Vietnam, because

no additional avian influenza A viruses could be isolated

from these geese. Three different viruses were identified as

the ancestors of the different genes found in DkNCVD1

(Fig. 1a–e; Table 2). The PB2, PB1, NP and NA genes of

DkNCVD1 were derived from A/teal/China/2978.1/02-like

(H5N1) virus (Table 2). The ancestor for the HA, M and NS

genes of DkNCVD1 was an A/quail/Shantou/3846/02-like

H5N1 virus (Fig. 1a, e; Table 2). The PA gene of

DkNCVD1 virus had a more distant ancestor, namely A/

partridge/Shantou/2886/03-like (H5N1) virus, showing

97% sequence homology (Fig. 1c; Table 2).

The identification of contemporary isolates from the

Guangxi and Shantou provinces of southern China as the

progenitors of the Vietnamese goose and duck viruses

characterized in this study suggests a possible virus intro-

duction from southern China to northern Vietnam [7, 24, 25,

40]. Phylogenetic analysis suggested that the genome of

LPAI Dk342 (H5N2) virus had multiple ancestors among

viruses from aquatic birds (Fig. 1a–e; Table 2). The H5 HA

and N2 NA descended from an A/duck/Primorie/2633/01-

like (H5N2) virus (98.0 and 98.6% homology, respectively)

[22]. The internal genes of Dk342 (H5N2) virus were derived

from aquatic-bird influenza viruses from southern China and

the Hokkaido region of Japan (Fig. 1b–e; Table 2).

The phylogenetic analysis of the genomes of LPAI duck

H9N3 viruses isolated from LBMs in Hanoi, Vietnam, in

2001 suggested that these viruses were reassortants and

derived their genes from multiple subtypes of avian influ-

enza viruses from aquatic birds and domestic poultry in

southern China, Hong Kong, Japan or Europe (Fig. 1b–e;

Table 2). None of the genes from Vietnamese LPAI H9N3

viruses appeared to be immediate ancestors of the HPAI

H5N1 viruses responsible for causing fatal H5N1 human

infections in Vietnam in late 2003. The HA of Dk68 and

Dk340 H9N3 virus isolates showed highest nucleotide

sequence homology with H9 HA of A/duck/Hokkaido/9/99

(H9N2) virus (96.98 and 98.73%, respectively). The N3

NA of Dk68 and Dk340 viruses were most closely related

to A/mallard/Italy/208/00 (H5N3) virus (96.88 and 95.83%

Fig. 1 Phylogenetic analysis of the genes of influenza A viruses

isolated from geese and ducks in Hanoi, Northern Vietnam, in 2001

and 2003. Phylogenetic trees were generated using neighbor-joining

analysis with the maximum composite model in the MEGA 4.0

program [33]. The reliability of the trees was assessed by bootstrap

analysis with 10,000 replicates, and the bootstrap values C60% are

indicated at tree nodes. The scale bar on each tree indicates

nucleotide substitution/site. Vietnam viruses from this study are

shown in bold font. The H5N1 viruses responsible for causing fatal

human infections in Vietnam in 2004 are shown in bold type and are

underlined. The outgroup virus isolates used for the different gene

segment trees are indicated in parentheses, H5 HA tree (A/tern/South

Africa/61); PB1 and PA tree (A/equine/London/1416/73); NP and M

tree (A/equine/Prague/1/56)

b

A/duck/Hong Kong/Y280/97A/chicken/Guangdong/10/00

A/duck/Hong Kong/7/75A/duck/Hong Kong/Y439/97

A/duck/Hokkaido/49/98

100

99 A/duck/Hokkaido/49/98Achicken/Germany/R28/03

A/duck/Fujian/19/00A/duck/Vietnam/68/01 (H9N3)

A/duck/Nanchang/4-165/00A/guinea fowl/Hong Kong/38/02

A/duck/Vietnam/342/01 (H5N2)A/d k/Ji i/1742/03

99

84A/duck/Jiangxi/1742/03

A/duck/Vietnam/340/01 (H9N3)A/chicken/Korea/06/96

A/quail/Hong Kong/G1/97A/chicken/Guangxi/9/99

A/Hong Kong/2108/03A/teal/Hong Kong/W312/97

8268

100

99

A/Hong Kong/156/97A/chicken/Hong Kong/G9/97

A/goose/Guangdong/3/97A/goose/Guangdong/1/96

A/duck/Hong Kong/2986.1/00A/chicken/Hong Kong/715.5/01

A/duck/Guangxi/35/01

85

100

100

92

100

A/duck/Guangxi/35/01A/duck/Guangxi/22/01A/goose/Vietnam/113/01 (H5N1)A/goose/Vietnam/324/01 (H5N1)

A/duck/Zheijang/52/00A/chicken/Hong Kong/YU777/02A/duck/Guangxi/50/01

A/ hi k /H K /96 1/02

9099

77

10066

A/chicken/Hong Kong/96.1/02A/duck/Vietnam/NCVD1/03 (H5N1)

A/teal/China/2978.1/02A/chicken/Vietnam/NCVD8/03A/duck/Guangxi/2291/04

A/duck/China/E319-2/03A/duck/Vietnam/568/05

7586

0.02

A/chicken/Shantou/4231/03A/chicken/Korea/ES/03A/egret/Hong Kong/7573/02

A/Hong Kong/213/03A/duck/Hong Kong/821/02

A/Vietnam/3062/04 (H5N1)A/Vietnam/1203/04 (H5N1)

99

93A/Vietnam/1203/04 (H5N1)

A/chicken/Vietnam/27/03A/Vietnam/1194/04 (H5N1)

A/chicken/Vietnam/37/04A/mallard duck/Vietnam/133/0564

92

B: PB1

Fig. 1 continued

H5N1 avian influenza viruses from Vietnam 1253

123

A/Hong Kong/156/97A/teal/Hong Kong/W312/97A/quail/Hong Kong/G1/97

A/Hong Kong/1074/99A/duck/Hong Kong/Y280/97

98

100

A/chicken/Hong Kong/G9/97A/chicken/Guangdong/6/97

A/duck/Anyang/AVL-1/01A/duck/Fujian/1/02

A/pheasant/Hong Kong/sv674-15/02A/duck/Hong Kong/7/75

A/duck/Hokkaido/49/98

100100

100

A/duck/Hokkaido/447/00A/duck/Vietnam/68/01 (H9N3)

A/chicken/Otiva/8/04A/duck/Vietnam/340/01 (H9N3)

A/chicken/Guangdong/174/04A/duck/Hokkaido/84/02

A/turkey/Italy/4169/9983

9999

A/duck/Vietnam/342/01 (H5N2)A/mallard/Italy/37/02

A/chicken/Korea/323/96A/swine/Shandong/2/03A/goose/Guangdong/1/96

A/environment/Hong Kong/437-10/99A/chicken/Hong Kong/YU 822.2/0195

100

8491

100

A/duck/Shanghai/08/01A/duck/Guangxi/35/01

A/duck/Vietnam/NCVD1/03 (H5N1)A/partridge/Shantou/2886/03

A/duck/Guangxi/22/01A/goose/Vietnam/113/01 (H5N1)A/goose/Vietnam/324/01 (H5N1)

95

98

100

99 69

97

A/swine/Fujian/1/03A/swine/ Fujian/F1/03

A/chicken/Hong Kong/61-9/02 A/teal/China/2978.1/02

A/chicken/Vietnam/NCVD8/03A/goose/Guangxi/914/04

A/duck/China/E319-2/03

82100

99

94

10076

0.02

A/chicken/Fujian/1042/05A/brown headed gull/ 3/05A/chicken/Guangxi/604/05

A/Hong Kong/213/03A/egret/Hong Kong/7572/03

A/duck/Hong Kong/821/02A/Vietnam/1194/04 (H5N1)

99

9473

84

68A/Vietnam/1204/04 (H5N1)A/Vietnam/1203/04 (H5N1)

A/duck/Vietnam/11/04A/quail/Vietnam/36/04A/chicken/Vietnam/24/04

A/duck/Vietnam/367/05

80

94

100

C: PAFig. 1 continued

1254 S. J. Jadhao et al.

123

A/duck/Hong Kong/7/75A/chicken/Hong Kong/G9/97A/duck/Hong Kong/Y280/97

A/chicken/Hong Kong/FY150/01A/wild Duck/Shantou/4808/01

99

99

75

A/teal/Hong Kong/W312/97A/quail/Hong Kong/G1/97A/Hong Kong/1074/99A/Hong Kong/156/97

A/Hong Kong/483/97A/goose/Guangdong/1/96

A/duck/Hong Kong/ww461/00z

97

93

100

A/duck/Hong Kong/2986.1/00A/goose/Hong Kong/385.5/00

A/goose/Hong Kong/761/01A/goose/Vietnam/113/01 (H5N1)A/goose/Vietnam/324/01 (H5N1)

A/duck/Guangxi/22/01A/duck/Guangdong/22/02

99

96

66

91

99

99

/duc /Gua gdo g/ /0A/chicken/Hong Kong/YU822.2/01

A/aquatic bird/Hong Kong/399/99A/duck/Guangdong/40/00A/duck/Vietnam/342/01 (H5N2)

A/duck/Vietnam/340/01 (H9N3)A/Netherlands/33/03A/Netherlands/219/03

100

10099

76

9993

A/Netherlands/219/03A/chicken/Germany/R28/03

A/chicken/Nanchang/7-010/00A/quail/Nanchang/7-026/00

A/duck/Vietnam/68/01 (H9N3)A/turkey/Italy/220158/02/duck/Hong Kong/Y439/97

A/aquatic bird/Hong Kong/603/98

100

9387

8868 A/aquatic

A/duck/Zheijang/52/00A/teal/China/2978.1/02A/duck/Vietnam/NCVD1/03 (H5N1)

A/chicken/Vietnam/NCVD8/03A/duck/Guangxi/2291/04A/chicken/Shantou/4231/03A/chicken/Guangdong/174/04

8166

10084

9868

85 A/chicken/Guangdong/174/04A/bar headed goose/Qinghai/ 5/05

A/Hong Kong/213/03A/duck/Hong Kong/821/02

A/egret/Hong Kong/7572/03A/Vietnam/1194/04 (H5N1)

A/Vietnam/1203/04 (H5N1)

85

100

9868

68

A/duck/Guangxi/50/01A/duck/Guangdong/173/04

A/duck/China/E3192/03A/duck/Vietnam/568/05

A/goose/Guangxi/345/0576

71

680.02 A/Vietnam/3062/04 (H5N1)

D:NPFig. 1 continued

H5N1 avian influenza viruses from Vietnam 1255

123

A/chicken/Hong Kong/G9/97A/duck/Hong Kong/Y280/97

A/quail/Hong Kong/G1/97A/Hong Kong/156/9797

9999

A/teal/Hong Kong/w312/97A/Hong Kong/2108/03

A/duck/Hong Kong/7/75A/chicken/Korea/06/96

A/duck/Hong Kong/Y439/97A/duck/Primoriae/2633/01

A/duck/Vietnam/342/01 (H5N2)A/duck/Hong Kong/86/76

99

99

80

g gA/duck/Vietnam/68/01 (H9N3)

A/duck/Shantou/5048/01A/duck/Hokkaido/49/98

A/mallard/France/2525/01A/duck/Vietnam/340/01 (H9N3)

A/mallard/Netherlands/12/00A/wild duck/Shantou/988/00A/mallard/Bavaria/1/05

99

60

65

A/mallard/Bavaria/1/05A/wild duck/Shantou/1737/00

A/goose/Guangdong/1/96A/goose/Guangdong/3/97

A/duck/Anyang/AVL-1/01A/guinea fowl/Hong Kong/38/02

A/duck/Hong Kong/5734/01A/duck/Guangdong/22/02

A/chicken/Hong Kong/YU562/01

99

99

9971

A/chicken/HongA/duck/Guangxi/35/01A/duck/Hong Kong/3805/01

A/duck/Hong Kong/2986.1/00A/duck/Guangxi/22/01

A/goose/Vietnam/324/01 (H5N1)A/goose/Vietnam/113/01 (H5N1)

A/chicken/Guangdong/174/04

85

90

64

64

88

98 A/chicken/Vietnam/NCVD8/03A/teal/China/2978.1/02

A/duck/Vietnam/NCVD1/02 (H5N1)A/duck/Guangxi/2291/04

A/goose/Guangxi/914/04A/duck/Vietnam/568/05

A/chicken/Fujian/1042/05A/bar headedgoose/Qinghai/5/05

99

98

64

0.02

A/duck/Hong Kong/821/02A/egret/Hong Kong/757.3/02

A/Hong Kong/213/03A/chicken/Hong Kong/YU22/02

A/chicken//Shantou/4231/03A/duck/China/E319-2/03A/chicken/Indonesia/11/03

A/Vietnam/1203/04 (H5N1)

76

A/Vietnam/1203/04 (H5N1)A/Vietnam/1194/04 (H5N1)

A/mallard duck/Vietnam/133/05A/chicken/Vietnam/37/04A/Vietnam/3062/04 (H5N1)A/chicken/Vietnam/27/03

A/chicken/Vietnam/C58/04

64

E:MatrixFig. 1 continued

1256 S. J. Jadhao et al.

123

nucleotide homology, respectively) (phylogenetic tree not

shown). The M gene of Dk340 was most closely related to

A/mallard/Netherlands/12/00 (H7N3), and the NP gene

showed highest nucleotide sequence homology (Table 2)

with the A/Netherlands/33/03 (H7N7) and A/Netherlands/

219/03 (H7N7) viruses, which are known to have caused

human infection in the Netherlands in 2003 [10, 17].

Therefore, the NP and M gene phylogenies indicate that

Vietnamese Dk340 (H9N3) virus, A/mallard/Netherlands/

12/00 (H7N3) and A/Netherlands/33/03 (H7N7) virus had

similar ancestors for these two genes. Since Vietnam and

the the Netherlands are located on different continents, a

role of migratory birds and virus reassortments for inter-

continental spread of the progenitors of the NP and M gene

is suggested. The NS gene segment of the H9N3 viruses

belonged to allele B similar to that of HPAI A/goose/

Guangdong/1/96 (H5N1) virus. The genotyping of the H5

subtype viruses using the FluGenome tool [21] showed that

Dk68, a Vietnamese duck-origin H9N3 virus isolate

acquired its PB2 gene from a different lineage than the

H5N1 viruses (Table 1). Phylogenetically, the H5 HA gene

of the HPAI Gs113 and Gs324 viruses belonged to phylo-

genetic clade 3, whereas DkNCVD1 virus belonged to

phylogenetic clade 5 (Fig. 1a). Unlike H5N1 viruses bear-

ing clade 0, 1, 2 or 7 H5 HA, viruses with clade-3 and -5 HA

have not been reported to infect people to date [1, 11].

Molecular characterization

Hemagglutinin cleavage site and receptor binding domains

The H5 HA from geese and a duck H5N1 virus isolates

analyzed in this study have the polybasic amino acids motif

at the HA0 cleavage site, a characteristic of the HPAI

viruses in poultry (Fig. 2). The H5 HA of Gs113 and

Gs324 viruses possessed novel polybasic site, PRI-

ERRRKKR/GLF, with arginine (R) at the -9 position

(counting from the C terminus of HA1) instead of Q, which

is commonly observed in H5N1 HA cleavage sites of HPAI

viruses. The H5 HA cleavage site (PQREIRRKKR) of

DkNCVD1 H5N1 virus was identical to that of A/teal/

China/2978.1/02 [20]. The HA cleavage site sequence

PQRETR/GLF of DkVn342 virus was characteristic of

LPAI H5 virus and identical to that of A/duck/Primorie/

2633/01 (H5N3) virus. The H9 HA protein of Dk68 and

Dk340 H9N3 viruses exhibited connecting peptide

sequences that differed by a single amino acid at position

-2, [Dk68 (PAASSR) versus Dk340 (PAASGR)]. The

analysis of the available 948 H9 HA sequences of H9 virus

isolates from avian and mammalian species in the NCBI

GenBank indicated that although the significance is not

obvious, Serine at position -2 in the PAASSR/GLF

cleavage site is unique to the Dk68 H9 virus.Ta

ble

2H

igh

est

nu

cleo

tid

ese

qu

ence

ho

mo

log

yo

fH

5N

1v

iru

sg

enes

fro

mV

ietn

amto

the

infl

uen

zaA

vir

use

sse

qu

ence

sav

aila

ble

inG

enB

ank

Vir

us

PB

2P

B1

PA

HA

NP

NA

MN

S

gs1

13

(H5

N1

)

dk

/Gx

/22

/01

(H5

N1

)(9

9.0

0)a

dk

/Gx

/22

/01

(H5

N1

)(9

9.6

9)

dk

/Gx

/22

/01

(H5

N1

)(9

9.5

3)

dk

/Gx

/22

/01

(H5

N1

)(9

8.8

1)

dk

/Gx

/22

/01

(H5

N1

)

(98

.54

)

dk

/Gx

/22

/01

(H5

N1

)

(99

.08

)

dk

/HK

/29

86

.1/0

0

(H5

N1

)(9

9.3

9)

dk

/HK

/38

0.5

/01

(H5

N1

)(9

8.1

3)

gs3

24

(H5

N1

)

dk

/Gx

/22

/01

(H5

N1

)(9

9.3

5)

dk

/Gx

/22

/01

(H5

N1

)(9

9.7

7)

dk

/Gx

/22

/01

(H5

N1

)(9

9.4

8)

dk

/Gx

/22

/01

(H5

N1

)(9

8.9

3)

dk

/Gx

/22

/01

(H5

N1

)

(99

.13

)

dk

/Gx

/22

/01

(H5

N1

)

(99

.15

)

dk

/HK

/29

86

.1/0

0

(H5

N1

)(9

9.3

0)

dk

/HK

/38

0.5

/01

(H5

N1

)(9

8.1

3)

dk

NC

VD

1

(H5

N1

)

teal

/Ch

ina/

29

78

.1/

02

(H5

N1

)

(99

.30

)

teal

/Ch

ina/

29

78

.1/

02

(H5

N1

)

(99

.06

)

par

trid

ge/

ST

/

28

86

/03

(H5

N1

)(9

7.0

)

qu

ail/

Sh

anto

u/

38

46

/02

(H5

N1

)

(99

.14

)

teal

/Ch

ina/

29

78

.1/0

2

(H5

N1

)(9

9.6

0)

teal

/Ch

ina/

29

78

.1/0

2

(H5

N1

)(9

9.6

2)

qu

ail/

Sh

anto

u/3

84

6/0

2

(H5

N1

)(9

9.5

6)

qu

ail/

Sh

anto

u/

38

46

/02

(H5

N1

)

(99

.51

)

dk

34

2

(H5

N2

)

dk

/Tai

wan

/WB

29

/

99

(H6

N1

)

(98

.25

)

dk

/Gx

/22

/01

(H5

N1

)(9

6.7

0)

mal

lard

/Ita

ly/3

7/

02

(H5

N3

)

(97

.54

)

dk

/Pri

mo

rie/

26

21

/

01

(H5

N2

)

(98

.62

)

aqu

atic

bir

d/H

on

g

Ko

ng

/39

9/9

9(H

3N

8)

(97

.86

)

Du

ck/E

aste

rnC

hin

a/

72

9/0

3(H

6N

2)

(98

.37

)

dk

/Ho

kk

aid

o/8

6/0

1

(H2

N3

)(9

8.0

5)

mal

lard

/Ita

ly/4

3/0

1

(H7

N3

)(9

7.9

7)

dk

68

(H9

N3

)

ck/H

K/3

1.2

/02

(H5

N1

)(9

8.3

9)

dk

/Nan

chan

g/4

-

16

5/0

0(H

4N

6)

(96

.99

)

dk

/Ho

kk

aid

o/8

4/

02

(H5

N3

)

(98

.20

)

dk

/Ho

kk

aid

o/9

/99

(H9

N2

)(9

6.9

8)

ck/N

anch

ang

/7-0

10

/00

(H3

N6

)(9

7.7

2)

mal

lard

/Ita

ly/2

08

/00

(H5

N3

)(9

6.8

8)

dk

/Mo

ng

oli

a/5

4/0

1

(H5

N2

)(9

9.6

9)

dk

/Ho

kk

aid

o/1

20

/

01

(H6

N2

)

(99

.02

)

dk

34

0

(H9

N3

)

dk

/Zh

ejia

ng

/11

/00

(H5

N1

)(9

6.8

6)

dk

/Jia

ng

xi/

17

42

/03

(H7

N7

)(9

8.2

6)

dk

/Ho

kk

aid

o/4

47

/

00

(H5

N3

)

(98

.20

)

dk

/Ho

kk

aid

o/9

/99

(H9

N2

)(9

8.7

3)

Th

eN

eth

erla

nd

s/3

3/0

3

(H7

N7

)(9

7.7

2)

mal

lard

/Ita

ly/2

08

/00

(H5

N3

)(9

5.8

3)

mal

lard

/th

e

Net

her

lan

ds/

12

/00

(H7

N3

)(9

9.1

9)

dk

/Ho

kk

aid

o/4

47

/

00

(H5

N3

)

(99

.39

)

aO

nly

the

infl

uen

zav

iru

ses

wit

hh

igh

est

deg

ree

of

nu

cleo

tid

ese

qu

ence

ho

mo

log

yb

ased

on

the

NC

BI

Gen

Ban

kn

ucl

eoti

de

BL

AS

Tse

arch

anal

ysi

sar

ein

clu

ded

inth

eta

ble

H5N1 avian influenza viruses from Vietnam 1257

123

Fig. 2 Alignment and

comparison of HA amino acid

sequence of representative

H5N1 poultry viruses from

Vietnam in 2001 and 2003 and

in 2004–2005. The H5N1 virus

isolates from fatal human cases

are shown in bold type. Amino

acid sequences with a greenbackground represent potential

glycosylation sites, yellowindicates loss of glycosylation,

light blue indicates residues

involved in the receptor-binding

site, red/brick represents HA

sequence at the cleavage site,

and violet shows substitutions

among genotypes/lineages. A

consensus H5 HA amino acid

sequence was generated for

viruses known to have infected

humans in Vietnam during

2003–2005 (color figure online)

1258 S. J. Jadhao et al.

123

As shown in Table 3, the receptor-binding pocket of the

HPAI H5N1 Gs113, Gs324, DkNCVD1 and LPAI H5N2

Dk342 viruses retained amino acid residues that are typical

for binding to the avian cell receptor (a-2, 3-NeuAcGal)

[13, 30, 43]. The Vietnamese HPAI H5N1 Gs113, Gs324,

and DkNCVD1 viruses and the LPAI Dk342 H5N2 virus

characterized in this study possessed 6 potential glycosyl-

ation sites, at positions 12, 13, 25, 167 288, and 486 (H5

numbering). These viruses lacked a potential glycosylation

motif at position 154–156, similar to A/Hong/Kong/156/

97, A/goose/Guangdong/1/96 and A/Hong Kong/213/03

viruses [12, 32, 42]. The H5N1 viruses that circulated in

poultry and infected people in Vietnam in 2004–2005,

unlike Vietnamese 2001 and 2003 virus isolates possessed

an additional glycosylation site at position 154–156 [1, 20].

NA structural features

The stalk region of N1 NA genes of the HPAI Gs113,

Gs324 and DkNCVD1 viruses did not undergo the dele-

tions observed in most other HPAI H5N1 viruses. The N1

NA of Gs113 and Gs324 viruses lacked potential glyco-

sylation sites at positions 53 and 237 and thus had a total of

five potential sites. In contrast, the N1 NA of DkNCVD1

virus had seven potential glycosylation sites. The NA gene

products of Vietnamese goose and duck H5N1 isolates had

a histidine (H) at position 274, indicating susceptibility to

Oseltamivir [37]. Likewise, the N2 NA of Dk324 H5N2

virus possessed E119 and R292 amino acid residues,

indicating that the commonly mutated residues in NA-

inhibitor-resistant viruses were not present [35, 41]. No

mutations associated with resistance to the NA inhibitor

drug were found in the NA of these 2001 and 2003 avian

influenza virus isolates from Vietnamese waterfowl.

M2 protein

The M2 ion channel protein is a target of adamantane

influenza antiviral drugs. The Vietnamese 2001 and 2003

H5N1, H5N2 and H9N3 virus isolates analyzed in this

study lacked the typical amino acid mutations, which

include, L26F, V27A, A30T/S, S31N, and G34E, associ-

ated with resistance to the adamantane class of influenza

antivirals [4, 15, 33].

Molecular features of internal genes associated

with virulence

The presence of lysine at position 627 in the PB2 gene has

been reported to influence the host range and confer

increased virulence for some HPAI H5N1 viruses in the

mouse model [14, 29, 31]. Three HPAI H5N1 viruses,

Gs113, Gs324 and DkNCVD1, and LPAI Dk342 (H5N2)

and Dk68 (H9N3) virus had a glutamic acid (E) at position

627 of PB2. Interestingly, the H9N3 isolate Dk340 had a

lysine (K) at position 627. A unique seven-amino-acid

deletion (position 77–83) in the NS1 protein was observed

in Gs113 and Gs324 H5N1 viruses; however, only five-

amino-acid deletions (position 80–84) were observed for

DkNCVD1 virus. Deletion of several amino acids in the

NS1 gene has been observed more frequently in avian

influenza viruses in recent years, a feature of possible

Table 3 Molecular signatures in the genes of H5N1, H5N2 and H9N3 avian influenza viruses isolated from geese and ducks in live-bird markets

of Hanoi, Vietnam, in 2001 and 2003

Virus isolate HA receptor-binding residues NA M2 PB2 NS1

129 212 217 222 224 274 279 26 27 30 31 34 627 92

A/goose/Guangdong/1/96 (H5N1)a S E P Q G H N L V A S G E D

A/Hong Kong/156/97 (H5N1)a S E P Q G H N L V A S G E D

A/Vietnam/1194/04 (H5N1)a L R S Q G H N I V A N G E D

A/Vietnam/1203/04 (H5N1)a L R S Q G H N I V A N G E D

A/Vietnam/1204/04 (H5N1)a L R S Q G H N I V A N G E D

A/goose/Vietnam/113/01 (H5N1)a S K S Q G H N L V A S G E D

A/goose/Vietnam/324/01 (H5N1)a S K S Q G H N L V A S G E D

A/duck/Vietnam/NCVD1/03 (H5N1)a S K S Q G H N L V A S G E D

A/duck/Vietnam/342/01 (H5N2)a S E P Q G ND ND L V A S G E D

A/duck/Vietnam/68/01 (H9N3)b Y V I Q G ND ND L V A S G E D

A/duck/Vietnam/340/01 (H9N3)b Y V I Q G ND ND L I A S G K D

The H5N1 virus isolates that caused fatal human illness are shown in bold type

ND not donea H5N1 HA amino acids are numbered according to H5 numberingb H9N3 HA amino acids are numbered as per H3 numbering

H5N1 avian influenza viruses from Vietnam 1259

123

adaptation of these viruses to poultry [5, 44, 45]. The LPAI

Gs342 H5N2 and Dk68 H9N3 viruses did not show a

deletion in the NS1 gene. The NS1 protein of domestic-

waterfowl HPAI H5N1 and LPAI H5N2 and H9N3 viruses

analyzed in this study possessed aspartic acid (D) at posi-

tion 92. Pathogenesis studies using A/Hong Kong/156/97

(H5N1) 9 A/Puerto Rico/8/34 (H1N1) reassortant virus

carrying the A/Hong Kong/156/97 (H5N1) NS1 gene with

glutamic acid (E) at position 92 have shown enhanced

virulence in pigs [27].

In summary, our genetic analysis suggests that the

HPAI H5N1 viruses isolated from domestic waterfowl in

Hanoi, northern Vietnam, in 2001 and 2003 are not the

nearest progenitors of the HPAI H5N1 viruses that

caused widespread outbreaks in poultry and human

infections in Vietnam beginning in late 2003. The

phylogenetic analyses indicates that the progenitors of

these Vietnamese 2001 goose and 2003 duck HAPI

H5N1 virus isolates were most closely related to viruses

contemporarily prevalent among domestic waterfowl in

southern China. HPAI H5N1 viruses with HA genes

belonging to clade 3 and clade 5 have not been found to

infect people in Vietnam or elsewhere to date. Our data

suggest that clade-1 HPAI H5N1 viruses that caused

widespread H5N1 outbreaks in poultry and human

infections beginning in late 2003 did not evolve from

these 2001 goose and 2003 duck H5N1 viruses that were

detected in Hanoi, northern Vietnam. The phylogenetic

analysis and epidemiological data are consistent with the

notion that HPAI H5N1 clade 1 viruses were introduced

independently into Vietnam after 2002 [23, 40]. More

recently, clade 2.3.4 H5N1 viruses that were prevalent in

southern China have been introduced into northern

Vietnam, further underscoring the role of domestic

waterfowl and poultry movement in the spread of variant

H5N1 viruses [25].

Acknowledgments We thank the National Institute of Hygiene and

Epidemiology and National Center for Veterinary Diagnosis, Hanoi,

Vietnam, for invaluable leadership in conducting of outbreak inves-

tigations. SJJ and DCN were supported by International Emerging

Infectious Disease Fellowship at the US Centers for Disease Control

and Prevention and administered by the Association of Public Health

Laboratories, USA. We thank P. Rivailler for sequence data annota-

tion and management. The findings and conclusions in this report are

those of the authors and do not necessarily represent the views of the

Centers for Disease Control and Prevention or the Agency for Toxic

Substances and Disease Registry.

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